Human Immunodeficiency Virus (HIV) is the etiological agent of acquired human immune deficiency syndrome (AIDS) and related disorders. HIV-1/HIV-2 are RNA viruses of the Retroviridae family and exhibits the 5′ LTR-gag-pol-env-LTR 3′ organization of all retroviruses. The integrated form of HIV, known as the provirus, is approximately 9.8 Kb in length. Each end of the viral genome contains flanking sequences known as long terminal repeats (LTRs). The HIV genes encode at least nine proteins and are divided into three classes; the major structural proteins (Gag, Pol, and Env), the regulatory proteins (Tat and Rev); and the accessory proteins (Vpu, Vpr, Vif and Nef).
The env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gp160) and then cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gp120) and the transmembrane 41-kDa envelope glycoprotein (gp41). Gp120 and gp41 remain associated and are displayed on the viral particles and the surface of HIV-infected cells. Gp120 binds to the CD4/chemokine receptor present on the surface of helper T-lymphocytes, macrophages and other target cells in addition to other co-receptor molecules. X4 (macrophage tropic) virus show tropism for CD4/CXCR4 complexes while a R5 (T-cell line tropic) virus interacts with a CD4/CCR5 receptor complex. After gp120 binds to CD4, gp41 mediates the fusion event responsible for virus entry. The virus fuses with and enters the target cell, followed by reverse transcription of its single stranded RNA genome into the double-stranded DNA via a RNA dependent DNA polymerase. The viral DNA, known as provirus, enters the cell nucleus, where the viral DNA directs the production of new viral RNA within the nucleus, expression of early and late HIV viral proteins, and subsequently the production and cellular release of new virus particles.
Gp41-mediated fusion is a complex process involving three essential components located in the ectodomain of the glycoprotein: an NH2-terminal fusion peptide, an NH2-terminal heptad repeat (“N-helix”) and a COOH-terminal heptad repeat (“C-helix”). The two heptad repeat regions (NH2:HR1; COOH: HR2) impart periodic hydrophobicity to the glycoprotein and are predictive of alpha-helical structures that interact with each other to form a fusogenic (i.e., fusion-active) conformation of gp41 called the “trimer-of hairpins,” a common structural motif involved in the fusion mechanism of many enveloped viruses. The trimer-of-hairpins structure is a bundle of six α-helices: three α-helices (formed by C-helix regions from three gp41 ectodomains) packed in an antiparallel manner against a central, three-stranded coiled-coil (formed by N-helix regions from three gp41 ectodomains). The fusion process progresses via the formation of a “pre-hairpin” conformation of gp41 that places the NH2-terminal fusion peptide near/in the target cell membrane, exposing the N-helix coiled-coil. The trimer-of-hairpins forms when three C-helices fold back to associate with the central, N-helix coiled-coil, drawing the viral and host cell membranes into close contact as a prelude to membrane fusion.
Effective treatment regimens for HIV infected individuals have become available recently. However, these drugs will not have a significant impact on the disease in many parts of the world and they will have a minimal impact in halting the spread of infection within the human population. One additional arm to add to the treatment arsenal would be an antibody that acts as an anti-viral agent for the treatment of HIV-associated disease. To the best of the inventors knowledge, there is currently no known isolated anti-viral antibody targeting the gp41 HR1 pre-hairpin with the ability to block HIV infection. It would also be advantageous to select for additional HIV antiviral agents and/or peptide vaccines for use in treating HIV infected individuals. Selection of an antibody targeting this region would allow for high throughput screening assays which would be useful in selecting new HIV antiviral agents.
U.S. Pat. No. 5,459,060 (issued 17 Oct. 1995), U.S. Pat. No. 6,008,044 (issued 28 Dec. 1999) and U.S. Pat. No. 6,983,504 (issued 4 Jul. 2000), each to J. P. Cotropia, disclose a human monoclonal antibody that interacts with the region around amino acids 602-611 of gp41.
U.S. Pat. No. 5,731,189 (issued 24 Mar. 1998 to Zolla-Pazuer and Gorny) discloses a series of gp41 specific human monoclonal antibodies that interact somewhere within a region from amino acids 579-663, depending upon the antibody.
U.S. Pat. Nos. 5,753,503 (issued 19 May 1998) and 5,831,034 (3 Nov. 1998), each to H. W. D. Katinger, disclose a series of human monoclonal antibodies raised against gp41. The '034 patent discloses hybridornas which produce 2F5 and 4E10 antibodies, which interact with epitopes downstream of the C-Heptad repeat of gp41.
U.S. Patent Application Publication US 2003/0118985 (published 26 Jun. 2003 in the name of Hunt et al.) discloses a human monoclonal antibody that interacts with the region around amino acids 649-672.
Louis et al. (2003, J. Biol. Chem. 278(22): 20278-20285) suggest that a fraction of a polyclonal sera raised against a covalent trimer of the gp41 trimeric coiled-coil region inhibits HIV envelope mediated cell fusion in vitro.
It would be of great import in the battle against AIDS to produce a therapeutic-based HIV anti-viral antibody targeting gp41 that possesses the ability to therapeutically reduce viral load levels within an infected individual, thus prolonging the asymptomatic phase of HIV infection. The present invention addresses and meets this needs by disclosing a class of neutralizing anti-HIV antibodies which interact with the HR1 region of the pre-hairpin, pre-fusion gp41 intermediate, and associated screening assays.